Spatial distributions of and species differences in 90Sr accumulation in marine fishes from the Fukushima coastal region.

Large amounts of radionuclides were released into the ocean during the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident. One of the released radionuclides, 90Sr, tends to accumulate in bone, and could conceivably have long-term negative impacts on fishes. Here, we investigated 90Sr radioactivity concentrations in the vertebrae of demersal fishes collected off the coast of Fukushima Prefecture in January and July 2014. High 90Sr radioactivity concentrations were detected in fish collected from central Fukushima (offshore of the FDNPP) and southern Fukushima (offshore of Iwaki City), but were almost hardly detected in northern Fukushima (offshore of Soma City). Additionally, 90Sr radioactivity concentrations were higher among rays (Okamejei kenojei and Hemitrygon akajei: from 1.1 ± 0.5 to 103.3 ± 15.1 mBq/g Ca) and soles (Cynoglossus joyneri and Paraplagusia japonica: from 18.5 ± 2.8 to 52.8 ± 11.3 mBq/g Ca) than among other species. Vertebral stable Sr/Ca ratios also differed by fish species. Ca concentrations were relatively constant among species, but Sr concentrations were higher among elasmobranchs and soles, indicating that differences in Sr/Ca might be related to differences in 90Sr accumulation in fish vertebrae.

Potential differences in chitin synthesis ability cause different sensitivities to diflubenzuron among three strains of Daphnia magna.

Ecotoxicity testing of crustaceans using Daphnia magna has been implemented in the chemical management systems of various countries. While the chemical sensitivity of D. magna varies depending on genetically different clonal lineages, the strain used in ecotoxicity tests, including the acute immobilization test (OECD TG202), has not been specified. We hypothesized that comprehensive gene expression profiles could provide useful information on phenotypic differences among strains, including chemical sensitivity. To test this hypothesis, we performed mRNA sequencing on three different strains (NIES, England, and Clone 5) of D. magna under culture conditions. The resulting expression profile of the NIES strain was clearly different compared to the profiles of the other two strains. Gene ontology (GO) enrichment analysis suggested that chitin metabolism was significantly enriched in the NIES strain compared to that in the England strain. Consistent with the GO analysis, evidence of high levels of chitin metabolism in the NIES strain were observed across multiple levels of biological organization, such as expression of chitin synthase genes, chitin content, and chitinase activity, which suggested that the different strains would exhibit different sensitivities to chemicals used to inhibit chitin synthesis. We found that among all strains, the NIES strain was more tolerant to diflubenzuron, a chitin synthesis inhibitor, with a 14-fold difference in the 48 h-EC50 value for the acute immobilization test compared to the England strain. The present study demonstrates that the differences among strains in chitin metabolism may lead to sensitivity difference to diflubenzuron, and serves as a case study of the usefulness of comprehensive gene expression profiles in finding sensitivity differences.

Functional analyses of chitinolytic enzymes in the formation of calcite prisms in Pinctada fucata.

The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals and the nacreous layer consisting of aragonite tablets. The calcite crystal of prisms contains small angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, to reveal the function of those organic matrices in calcium carbonate crystallization, calcites synthesized in chitin gel with or without chitinolytic enzymes were analyzed by using transmission electron microscope (TEM) and atom probe tomography (APT), showing ion clusters derived from chitin inside of a calcite and small angle grain boundaries at optimal chitinolytic concentration. Furthermore, we performed the experiment in which chitinase inhibitor was injected into a living P. fucata. Nanoindentation and electron back scattered diffraction (EBSD) show that mechanical properties and crystal orientation were changed. These results suggested that chitinolytic enzymes work cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer.

Evolution of Biomineralization Genes in the Prismatic Layer of the Pen Shell Atrina pectinata.

Molluscan shells are composed of calcium carbonates, with small amounts of extracellular matrices secreted from mantle epithelial cells. Many types of shell matrix proteins (SMPs) have been identified from molluscan shells or mantle cells. The pen shell Atrina pectinata (Pinnidae) has two different shell microstructures, the nacreous and prismatic layers. Nacreous and prismatic layer-specific matrix proteins have been reported in Pteriidae bivalves, but remain unclear in Pinnidae. We performed transcriptome analysis using the mantle cells of A. pectinata to screen the candidate transcripts involved in its prismatic layer formation. We found Asprich and nine highly conserved prismatic layer-specific SMPs encoding transcript in P. fucata, P. margaritifera, and P. maxima (Tyrosinase, Chitinase, EGF-like proteins, Fibronectin, valine-rich proteins, and prismatic uncharacterized shell protein 2 [PUSP2]) using molecular phylogenetic analysis or multiple alignment. We confirmed these genes were expressed in the epithelial cells of the mantle edge (outer surface of the outer fold) and the mantle pallium. Phylogenetic character mapping of these SMPs was used to infer a possible evolutionary scenario of them in Pteriomorphia. EGF-like proteins, Fibronectin, and valine-rich proteins encoding genes each evolved in the linage leading to four Pteriomorphia (Mytilidae, Pinnidae, Ostreidae, and Pteriidae), PUSP2 evolved in the linage leading to three Pteriomorphia families (Pinnidae, Ostreidae, and Pteriidae), and chitinase was independently evolved as SMPs in Mytilidae and in other Pteriomorphia (Pinnidae, Ostreidae, and Pteriidae). Our results provide a new dataset for A. pectinata SMP annotation, and a basis for understanding the evolution of prismatic layer formation in bivalves.

Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata.

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Structural and functional analyses of calcium ion response factors in the mantle of Pinctada fucata.

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.

Crystal defects induced by chitin and chitinolytic enzymes in the prismatic layer of Pinctada fucata.

Biomineralization, in which organisms create biogenic hard tissues, with hardness or flexibility enhanced by organic-inorganic interaction is an interesting and attractive focus for application of biomimetic functional materials. Calcites in the prismatic layer of Pinctada fucata are tougher than abiotic calcites due to small crystal defects. However, the molecular mechanism of the defect formation remains unclear. Here, chitin and two chitinolytic enzymes, chitinase and chitobiase, were identified as organic matrices related to for the formation of small crystal defects in the prismatic layer. Experiments with a chitinase inhibitor in vivo showed chitinase is necessary to form the prismatic layer. Analysis of calcite crystals, which were synthesized in a chitin hydrogel treated with chitinolytic enzymes, by electron microscopy and X-ray diffraction showed that crystal defects became larger as chitin was more degraded. These results suggest that interactions between chitin and calcium carbonate increase as chitin is thinner.